title: Evaluation of potential reference genes for qRT-PCR studies in human hepatoma cell lines treated with TNF-α.
abstract: In this study, the expression of eight candidate reference genes, B2M, ACTB, GAPDH, HMBS, HPRT1, TBP, UBC, and YWHAZ, was examined to identify optimal reference genes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in two human hepatoma cell lines, BEL-7402 and SMMC-7721, treated with tumor necrosis factor-α (TNF-α) for different time periods. The expression stability of these genes was analyzed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Results showed that TBP was the most stably expressed gene in BEL-7402 and SMMC-7721 cell lines under current experimental conditions, and that the optimal set of reference genes required for accurate normalization was TBP and HMBS, based on the pairwise variation value determined with geNorm. UBC and ACTB were ranked as the least stable genes by same algorithms. Our findings provide evidence that using TBP alone or in combination with HMBS as endogenous controls could be a reliable method for normalizing qRT-PCR data in human hepatoma cell lines treated with TNF-α.
authors.affiliation: Department of Pathology, School of Basic Medical Sciences, Central South University, Changsha, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Wu
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Zhong
authors.forename: Hailing
authors.initials: H
authors.lastname: Liu
authors.forename: Qiongqiong
authors.initials: Q
authors.lastname: He
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yang
authors.forename: Jifang
authors.initials: J
authors.lastname: Wen
authors.forename: Deyun
authors.initials: D
authors.lastname: Feng
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics; D000199:Actins / Q000235:genetics; D000465:Algorithms; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D035181:TATA-Box Binding Protein / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025801:Ubiquitin / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics
chemical_list: D048948:14-3-3 Proteins; D000199:Actins; D035181:TATA-Box Binding Protein; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; C572235:YWHAZ protein, human; D001613:beta 2-Microglobulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase; D011163:Hydroxymethylbilane Synthase
keywords: cytokine; human hepatoma cell lines; qRT-PCR; reference gene; tnf-α